Shan-Lu Liu, Ph.D. - US grants

DESCRIPTION (provided by applicant): In this R21 application, we aim to determine how the newly identified cellular restriction factors, known as interferon-inducible transmembrane (IFITM) proteins, modulate viral membrane fusion and entry, and in doing so, aid the development of novel antiviral therapeutics. The specific aims of this proposal are as follows. Aim 1: Test the hypothesis that IFITM proteins inhibit membrane fusion by preventing hemifusion. We will perform cell-cell and virion-cell fusion assays, including the use of single viral particle imaging technique, to determine the stages of the viral membrane fusion process blocked by IFITM proteins. Lipid analogs that modulate distinct stages of the membrane fusion process will be applied. We will explore the possibility that IFITM proteins restrict membrane fusion induced by cellular and developmental fusogens. Aim 2: Test the hypothesis that expression of IFITM proteins changes the lipid order of cell membranes and confers positive spontaneous curvature. We will perform fluorescence membrane labeling experiments to test the hypothesis that IFITM proteins cause the cell membrane to be more ordered, resulting in reduced fluidity and therefore less competency for fusion. We will also determine the effect of IFITM proteins on membrane curvature by reconstituting artificial liposomes with synthetic peptides corresponding to the functional domains in IFITM proteins. Cryo-EM tomography will be applied to directly visualize the curvature changes upon insertion of IFITM peptides into the artificial liposome vesicles. IFITM proteins are the first and so far only cellular restriction fators that are known to restrict viral membrane fusion and entry. Results from the proposed studies will provide novel insight as to how IFITM proteins restrict viral membrane fusion. These results will guide future comprehensive investigations into the biology of IFITM proteins, as well as their critical roles in restricting viral entry and infection.

DESCRIPTION (provided by applicant): Interferon (IFN) plays a central role in host intrinsic immunity to viral infection, the underlying mechanism of which remains poorly defined. Towards this goal, we have recently shown that interferon-induced transmembrane (IFITM) proteins profoundly inhibit the membrane fusion and infection of a number of enveloped viruses, including HIV-1. Interestingly, we found that, while human IFITM2 and IFITM3 impede HIV- 1 (BH10) entry, human IFITM1 impairs viral infectivity. Notably, the prolonged culture of BH10 HIV-1 led to the emergence of mutations in HIV-1 Env that render the virus resistant to IFITM1 inhibition, suggesting that IFITMs may functionally act on HIV-1 Env and diminish viral infectivity. The goal of this R01 project is to determine the mechanisms by which IFITM proteins inhibit distinct steps of HIV replication, as well as viral antagonisms. Aim 1 will address how IFITM proteins inhibit HIV-1 entry. We will use novel cell-cell fusion and single virus fusion techniques to test the hypothesis that both hemifusion and pore expansion are inhibited by IFITM2 and IFITM3. Aim 2 will focus on how IFITM proteins, especially IFITM1, diminish HIV-1 infectivity. We will test the central hypothesis that IFITM proteins are incorporated into HIV-1 particles and functionally inactivate HIV-1 Env activity. Aim 3 will characterize HIV-1 antagonisms against IFITMs, particularly the possible role of HIV-1 Env in this process. Collectively, results from this project will provide critical insights into the mechanisms of actio of IFITMs, and will aid in the development of novel antiviral agents against HIV-1 infection.

DESCRIPTION (provided by applicant): Entry of Ebolavirus (EBOV) into host cells is mediated by its sole glycoprotein, known as GP. The GP and its associated EBOV entry events possess many unusual features that provide novel insights into our fundamental understanding of viral entry. In this R21 project, we aim to elucidate how the newly identified cellular restricton factors, known as interferon-inducible transmembrane (IFITM) proteins, especially IFITM2, potently and specifically inhibit EBOV entry, and in doing so, aid the development of novel antiviral therapeutics. Aim 1: Establish a single virus fusion assay for EBOV and dissect the stages of membrane fusion inhibited by IFITM2. We will take advantage of the fact that EBOV GP can be efficiently incorporated into its virus-like particles (VLPs) formed by the VP40 matrix protein, and develop a single virus imaging and fusion system to determine how IFITM2 inhibits EBOV fusion in endolysosomes. Aim 2: Elucidate the molecular and biochemical mechanisms by which IFITM2 specifically inhibits EBOV GP-mediated entry. We will test the central hypothesis that IFITM2 profoundly inhibits EBOV entry by disturbing the triggering capability and/or the cholesterol transport activity of its intracellular receptor, Niemann-Pick C1 (NPC1). A series of biochemical and novel fluorescence lipid labeling techniques will be used to assess the effect of IFITM2 on cholesterol content, membrane fluidity, and conformational changes of EBOV GP. EBOV is a highly pathogenic filovirus that causes severe hemorrhagic fever in humans, with a fatality rate of up to 90%. Results from the proposed studies will provide critical novel insight into how IFITM2 restricts EBOV GP-mediated membrane fusion and entry, as well as advance our understanding of the general mechanism of IFITMs that block viral entry.

DESCRIPTION (provided by applicant): The long-term objective of our research is to better understand the mechanisms of viral entry and the possible implications for therapeutic interventions. To achieve these goals, we have recently studied the interferon-inducible transmembrane (IFITM) proteins that potently inhibit entry and infection of a wide range of viruses, including those of the highly pathogenic influenza A virus (IAV), SARS coronavirus, Ebolavirus (EBOV), and HIV-1. We showed that IFITM proteins profoundly inhibit cell-cell fusion induced by IAV HA, Semliki Forest virus (SFV) E1, and vesicular stomatitis virus (VSV) G proteins, which represent class I, II and III viral fusion proteins, respectively. Further experiments revealed that IFITMs block the creation of viral membrane hemifusion, which is consistent with their ability to decrease membrane fluidity. Interestingly, we observed that some viruses are more sensitive than others to inhibition by particular types of IFITMs, suggesting that IFITM-mediated restriction of viral entry can, in addition to broad inhibitions, be virus dependent The specific aims of this project are (1) to determine the molecular and biophysical mechanisms by which IFITMs inhibit viral membrane fusion, (2) to understand the molecular basis by which IFITM proteins change lipid properties and membrane mechanics, thereby inhibiting viral membrane fusion, and (3) to elucidate the molecular and cellular control mechanisms that govern the differential antiviral activities of IFITMs. Collectively, understanding the underlying mechanisms of IFITMs as proposed in this work will significantly advance our knowledge of IFN-mediated intrinsic immunity against viral entry. Results from the proposed experiments will aid the development of novel therapeutic agents against viral infections.

The majority of antiviral restriction factors are interferon (IFN) inducible, and are thus collectively referred to as IFN-stimulated genes (ISGs); many, however, are NOT directly regulated by IFN and remain poorly characterized. Notably, some of these cellular factors are known to modulate lipids and/or membrane properties, thereby disrupting the replication of HIV and other viruses. Two recent examples from this category are TIM (T- cell immunoglobulin and mucin domain) and SERINC (serine incorporator) family proteins, which directly interact with or possibly regulate the synthesis of phosphatidylserine (PS), thus inhibiting HIV release or infectivity. Interestingly, our preliminary data and two recent reports published in Nature showed that the lentiviral Nef proteins effectively antagonize the restriction by TIMs and SERINCs. Moreover, we have recently observed that SERINC proteins potentiate the ability of TIM-1 to block HIV-1 release and that SERINCs do this by stabilizing the TIM expression in the viral producer cells. In this application, we propose to test several novel hypotheses that address the possible link between TIM, SERINC, PS and Nef. Aim 1 will determine how HIV-1 Nef antagonizes TIM-mediated inhibition of viral release through modulating the synthesis and trafficking of TIM-1 and PS. Aim 2 will focus on understanding of the role of endogenous SERINC proteins in CD4+ T cells that regulates the TIM expression and stability, as well as in modulating lipids in the viral producer cell and viral particles, collectively contributing to the inhibition of HIV-1 release and replication. Aim 3 will define the molecular interplay between SERINC and TIM proteins in viral producer cells, and dissect how HIV-1 Nef protein down- modulates this process to promote HIV-1 production and infection. Results from the proposed experiments will provide novel and unified mechanistic insights into the interplay between TIM, SERINC and HIV Nef, and will enhance our understanding of virus-host interaction and AIDS pathogenesis.